The rapid worldwide proliferation of Methicillin-Resistant S. aureus (MRSA) poses a major threat to public health and produces infections that have proven to be more difficult to treat compared to Methicillin- Susceptible S. aureus (MSSA) infections. The reason for this is unknown, but reduced effectiveness of common antibiotics and prolonged inflammation likely contribute to the pathophysiology of infection, especially in the lung. While it is self-evident that MRSA may be resistant to common antibiotics, the source of enhanced or prolonged inflammation may or may not be related to perseverance of the bacteria. The cell wall of S. aureus (like all Gram-positive bacteria) is made up primarily of highly cross-linked peptidoglycan polymer, and recent work from our labs has suggested that degradation of the S. aureus peptidoglycan by phagocytes strongly enhances inflammatory responses by releasing pro-inflammatory intracellular components such as bacterial DNA. S. aureus actively modifies its peptidoglycan to make it resistant to degradation and suppresses inflammatory responses, and when this effect is inhibited, S. aureus induces greater cytokine production from macrophages in vitro and causes more immunopathology in mice. We have further found that sub-lethal alterations in cell wall peptidoglycan synthesis induced by antibiotics in S. aureus als causes macrophages to elicit a more powerful inflammatory response. MRSA becomes antibiotic-resistant through induced expression of PBP2A, a peptidoglycan synthesizing enzyme that is resistant to ?-lactam antibiotics. Expression of PBP2A, however, has been reported to cause alterations in peptidoglycan synthesis and structure, and we have now observed that this directly enhances inflammatory signaling in macrophages. Based on these findings, we come to the surprising hypothesis that specific antibiotics may actually cause immunopathology to become more pronounced during MRSA infection. We will test this hypothesis in two aims. In the first aim, we will examine directly the effects of PBP2A and antibiotics typically used in the clinic on the inflammatory capacity of S. aureus in vitro. Using cells from knockout mice we will identify the mechanisms underlying alterations in inflammatory capacity. In the second aim, we will move to in vivo mouse pneumonia models to determine the effects of antibiotics and PBP2A expression by MRSA on immunopathology. PUBLIC HEALTH RELEVANCE: Compared to Methicillin-Sensitive S. aureus (MSSA), Methicillin-Resistant S. aureus (MRSA) causes lung infections that appear more severe and are harder to treat. Here we investigate the hypothesis that cell wall active antibiotics and the gene encoding methicillin resistance both contribute to increased immunopathology. If our hypothesis is correct, our research could uncover an important cause of morbidity in MRSA pneumonia patients, and could pave the way for more effective ways to treat MRSA infections by addressing the damage caused by inflammation.